The present invention relates to coated polymer articles and processes for the production thereof and use of the article as chromatography medium. Specifically the invention relates to coated articles with the coating covalently bound to the polymer surface via pending double bonds of the polymer surface. An example of pending groups are residual vinyl groups remaining after polymerisation of vinyl monomers, such as divinyl benzene monomers.
In the context of the present invention the term xe2x80x9ccoatedxe2x80x9d/xe2x80x9ccoatingxe2x80x9d means that pending alken groups on the surface has been derivatized to introduce novel functionaities that may be adapted to two specific uses of the article (chromatographic support and support for oligopeptide and oligonucleotide synthesis). xe2x80x9cCoatxe2x80x9d thus has a somewhat different meaning compared to coating in the sense of physical adsorption and stabilization by crosslinking of an adsorbed layer (coat) within the layer and/or to the original surface.
The stationary phase (support, matrix) in liquid chromatography is composed of a porous matrix and is most commonly in bead form. A wide variety of materials are used for chromatography matrices, both inorganic and organic materials. The demands on the matrix are several. It should be chemically and physically stable and be able to withstand extreme pH conditions. The matrix should be rigid to allow high flow rates in columns packed with particles of small diameters. It must also be possible to produce particles with a broad range of porosities.
The surface characteristics of the matrix is important. By introduction of new chemical structures on the surface of the matrix it is possible to design stationary phases which interact more or less specifically with a particular molecule. In chromatographic separation of proteins in aqueous solution the matrix is usually hydrophilic. In reversed phase chromatography (RPC) for peptide or protein separation, silica gels have been used as chromatography medium. Silica gels are rigid enough to withstand high flow rates and different hydrophobicities are available (C4, C8, C18). However, silica gels are not stable above pH 8.0 and hence can not be cleaned by using alkali. In stead it is common to use rigid matrices of organic polymers e.g. polymethacrylate or polystyrene/polystyrene-divinylbenzene. Polystyrene-divinylbenzene particles are hydrophobic in aqueous solutions and large amounts of organic solvents have to be used to eluate the absorbed molecules. The selectivity is also limited. Due to the fact that there is only a small variation in hydrophobicity of these particles, the use of them is limited. In contrast the ratio hydrophobicity/hydrofilicity of silica particles can be varied by chemical reactions. There are a large variety in chemistry available for modification of the hydrofilic silica surface. To increase the possibilities to use particles of polystyrene-divinylbenzene and other hydrophobic polymers, it is recognized that the surface of the particles must be modified to be more hydrophilic before they can be used as chromatographic separation medium. Different methods have been suggested for hydrophilisation of the surface of particles based on hydrophobic polymers:
WO 91/11241 relates to a method of producing a hydrophilic coating on a hydrophobic surface by which a compound comprising a hydrophobic and a hydrophilic domain is adsorbed on the surface. The draw backs with this method are several. The polymer to be adsorbed has a high molecular weight and therefore the polymer will not spread out evenly in all pores. The control of the pore size distribution is then lost and some pores may not even be coated at all. As the polymer coating is only adsorbed it may loosen from the surface at contacting with non-polar solvents. Therefore the coating must be cross-linked.
WO 95/23022 discloses a method of covalently bonding a hydrophilic coating on a hydrophobic surface. An unsaturated polymer coating is grafted to the surface via the unsaturated groups on the surface. Also with this method there is the same problem of obtaining the coating evenly distributed on the porous surface of the matrix.
Various uses of vinyl groups pending on vinyl benzene polymers have been reviewed during the priority year by Hubbard et al (Reactive Functional Polymers 36 (1998) 1-16 and 17-30). The use of pending vinyl groups for immobilizing a catalyst on a macroporous polystyrene resin has been described by Faber et al (Reactive Polymers 11 (1989) 117-126. Modifying chromatographic vinyl-benzene based supports by routes other than via residual vinyls has been described by Sun et al (J, Chromatog, 522 (1990) 95-105). Still another route has been described by Moberg et al (Reactive Polymers 15 (1991) 25-35).
The object of the present invention is to obtain improved coated polymer articles and improved methods of coating a polymer article to change the surface characteristics of the article without the draw backs of known methods.
A further object of the invention is to produce polymer particles with suitable surface characteristics for chromatographic use.
It is a further object of the invention to provide a coating which may be derivatized to produce a wide range of different functional groups for use in different types of chromatography. The intention is to be able to achieve a large variety in both hydrophobicity/hydrophilicity and selectivity and to reduce the amount of solvent used in the elution.
The demands on supports to be used in the synthesis of oligonucleotides and oligopeptides with respect to the balance between hydrophobicity and hydrophilicity are less critical than for chromatographic matrices, although it remains that maintaining open pores is important in order to maintain high capacity of a given porous support. The primary object in this aspect of the invention is to provide a simple well-defined way of introducing a handle in form of a primary or secondary amino group into a polymer support based on a hydrophobic polymer.
The objects of the invention are achieved by the polymer articles and methods defined in the claims. According to the invention a coated polymer article is obtained, which article is characterized in that the coating is covalently bound to the polymer surface via double bonds of the polymer. The article has the structure:
xe2x80x83PLCHxe2x80x94CH2Y(R2)p
X(R1)p
where
P comprises the basic polymer structure of the article before coating and further groups
xe2x80x94Lxe2x80x94CH(xe2x80x94X(R1)p)xe2x80x94CH2(xe2x80x94Y(R2)p);
L is a part of a pending group projecting from the basic polymer and comprising the double bonds utilized for introducing xe2x80x94X(R1)p and xe2x80x94Y(R2)p. It may be a single covalent link to the basic polymer or a benzene ring.
X and Y are independently selected from halogens, in particular Br, and N, S and O.R1 is hydrogen, a straight, branched or cyclic alkyl or acyl group, such as C1-C10 alkyl, C1-C18 acyl, or R2 when X is N, O or S with particular emphasis for X equals Y equals O.
R2 is hydrogen, straight, branched or cyclic alkyl, such as C1-C18 alkyl, xe2x80x94R3(xe2x80x94NH2)q, alkylaryl or arylalkyl in which the alkyl part may contain 1-18 carbon atoms,
xe2x80x94R3(xe2x80x94NHxe2x80x94CR4xe2x95x90O)q, xe2x80x94CR4xe2x95x90O, or poly lower alkyloxy groups that may have been terminally acylated or alkylated (e.g. xe2x80x94((CH2)nxe2x80x94O)n.ORxe2x80x2 where n is an integer 2, 3 or 4 and nxe2x80x2 is an integer 2-100 and an H in CH2 may have been replaced with methyl and Rxe2x80x2 may be any one of the expressly mentioned R1 groups.
p=0 when Y=Br. p may be 1 when Y=O or S. p may be 2 when Y=N or S. p may be 3 for Y=N. In case there are more than one R2, they may be identical or different.
R3 is a straight, cyclic or branched alkyl, xe2x80x94Oxe2x80x94alkyl, hydroxyalkyl, phenylalkyl, with up to 18 carbon atoms in the alkyl part.
R4 is a straight, branched or cyclic alkyl group, such as C1-C18 alkyl.
q represents that one or more (1, 2, 3, 4 etc, i.e. 1-poly) hydrogens in a basic alkyl group R3 may have been replaced with a respective primary amino group (xe2x80x94NH2) or a respective xe2x80x94NHxe2x80x94CR4xe2x95x90O group.
In the above-mentioned alkyl and acyl groups, one or more hydrogens may be optionally substituted with an amino or a hydroxy or an alkoxy group and carbon chains may be optionally broken at one or more locations by an ether oxygen or an amino nitrogen, the proviso being that there are at most one atom selected among oxygen and nitrogen binding to one and the same sp3-hybridised carbon atom. Among particularly interesting alkyl groups with broken hydrocarbon chains may be mentioned those comprising repetitive alkylene oxide units, such as xe2x80x94CH2CH2xe2x80x94Oxe2x80x94, xe2x80x94CH2CH2CH2xe2x80x94Oxe2x80x94, xe2x80x94CH2CH(CH3)xe2x80x94Oxe2x80x94 etc.
According to a further aspect of the invention a method of producing a coated polymer article is provided, characterized in derivatizing double bonds linked to the basic polymer structure (P) of the article through the group L.
According to yet another aspect of the invention a coated polymer article according to the invention is used as chromatography medium or as a support for the solid phase synthesis of an oligopeptide or an oligonucleotide.
With the present invention it is possible to apply a coating to any polymer article having double bonds on the surface and thus to modify the surface in a predetermined manner. Preferably the method is used to manufacture chromatographic media. It was found that a wide range of chromatography media can by synthesised by introducing different chemical structures on a porous polymer surface. A controlled amount of a desired group and/or charge can be introduced on the surface. With the method according to the invention the coating, including the desired group and/or charge, is evenly spread out over the surface also in the pores and then the pore size distribution is kept intact. Besides, the pore size distribution can be varied by varying the length of the groups introduced on the surface. The method is suitable for the production of reversed phase chromatography (RPC) media with the same performance as silica media but without the draw backs of silica. New RPC media are prepared by introducing polar groups containing long alkyl or amide groups close to the surface. By varying the length of the alkyl and amide chains, different matrices with various hydrophobicities and pore size distributions can be synthesised. Also primary and secondary amino groups may be introduced which are essential for the use of support in the synthesis of oligonucleotides.
According to the invention the double bond of a pending group is transformed to an halohydrin or vicinal halide, i.e. xe2x80x94CHX1xe2x80x94CHY1xe2x80x94, where at least one of X1 and Y1 is a substituent selected from halogens, in particular bromine, and the remaining group of X1 and Y1 is hydroxy or alkoxy, or to an epoxide or to corresponding nitrogen or sulphur analogues.
Halogen may be introduced by addition of certain halogen containing compounds, with the preferred halogen being bromine, to the double bonds present on the polymer surface. Bromine can be introduced by known methods such as reaction with Br2 in a suitable solvents, or with Nxe2x80x94Brxe2x80x94 succinimide in a suitable solvent. The synthesis will now be illustrated by discussing the reaction with bromine. This does not exclude the use of other halogens, such as chlorine and iodine, which often undergo analogous reactions.
Preferably, in the first step of the method a polymer compound one or more double bonds of a polymer (Pxe2x80x94Lxe2x80x94CHxe2x95x90CH2 with P and L having the same meaning as above) is reacted with Br2 in a solution containing R1OH or a mixture of H2O and an organic solvent soluble in H2O, whereby a coated polymer article having the structure 
or a mixture of the structures: (1) and 
are obtained, where
R1 is hydrogen or a straight, branched or cyclic alkyl group containing oxygen atoms as defined above. Particularly R1 may be hydrogen or C1-C10 alkyl.
As mentioned above the method according to the invention can be used on polymer surfaces with free double bonds on the surface. As such polymer can be mentioned polymers from aromatic compounds containing vinylic groups. The aromatic compounds can be mono-functional, di- or poly- functional. The polymer should have a sufficient amount of di- or poly vinylic groups to result in a cross-linked structure. As suitable aromatic compounds can e.g. be mentioned styrene, divinylbenzene, vinylbenzene, vinylbenzylchloride, etylvinylbenzene, acetoxyvinylbenzene and other derivatives of vinylbenzene. In a preferred embodiment the basic polymer structure is a cross-linked polymer of styrene, divinylbenzene and ethylvinylbenzene or a polymer of divinylbenzene and ethylvinylbenzene. The pending groups are then benzene groups resulting from vinyl groups which have not been crosslinked. The structure can be described as follows: 
The free double bonds from the vinyl groups on the surface of the polymer are used for the reaction with bromine.
In preferred embodiments of the invention the coated articles are particles. Preferably the coated particles are prepared by coating uncoated particles-with a diameter of 1-200 xcexcm, preferably 3-50 xcexcm. The surface area of the uncoated particles lies within the range 1-1000 m2/g, preferably within 100-800 m2/g, and suitable pore volumes are from 0.1-3.0 ml/g. The content of residual vinyl groups in the beads are normally 0.3-7.7 mmol/g and should preferably be 0.3-3 mmole/g, most preferably 1-2 mmole/g.
In the first step of the invention the uncoated polymer article is contacted with bromine dissolved in the same solvent. Often the polymer article is in the form of particles which for coating purposes is suspended in the same solvent as bromine. In the reaction with bromine the choice of solvent is important for the outcome of the reaction. The reaction with bromine can result in a monobromide or in a dibromide or in a mixture thereof, as described above. Usually a mixture is obtained with a varying content of mono- and di-bromide. This content depends on the solvent used in the process. It is known that bromination in non-polar solvents like tetrachloromethane results in a high content of dibromide. In polar solvents like the lower alcohols, i.e. straight or branched C1-C10 alcohols, a high content of the monobromide is obtained. However, in water the amount of dibromide achieved is high due to the fact that bromine is partly soluble in water. If water is mixed with an organic solvent, soluble in water, and in which bromine dissolves, then the monobromide is obtained to a large extent. With longer alkyl groups the alcohols become more non-polar and then the amount of dibromide increases. Generally it can be said that if the polarity, expressed by the dielectric constant ∈, for the solvents or solvent mixtures is low, a high content of dibromide will be obtained. If ∈ is high the production of the monobromide will be favoured. In the present invention a high amount of the monobromide is preferred. Then, ∈ between 6 and up to about 78 is preferred. Solvents suitable for the invention are C1-C10 alcohols such as methanol, ethanol, 1-propanol, 1-butanol, 1-hexanol. If a mixture of water and an organic solvent is used, solvents like tetrahydrofuran, lower alcohols are suggested. In this latter case R in the formula above will be H. There are many reports on the bromination reaction of vinyl groups. The following references can be mentioned: Yates, K, McDonald, R. S, and Shapiro., S. A., (1973 ): J. Org. Chem. 38, 2465; J. H. Rolston and K. Yates, (1969 ): J. Am. Chem. Soc.91, 1469; R. C. Fahey, (1989 ): Topics in stereochemistry, Wiley, N.Y., p 280-286.
The reaction with bromine is suitably made at a temperature between xe2x88x9220-50xc2x0 C., preferably 0-20xc2x0 C. The amount of bromine used is suitably 0.1-10 moles bromine/mole vinyl, preferably 0.5-2 moles bromine/mole vinyl. The amount of monobromide obtained is also dependent of the bromine concentration. Concentrations of 0.05-0.5 moles/l solvent can be used, preferably 0.1-0.2 moles/l.
In the next step of the invention the brominated articles of the invention are reacted with a compound Y(R2)p whereby a coated polymer article having the structure 
or a mixture of the structures (3) and 
are obtained, where
Y=N, S, O, and R2, R3 and p have the same meaning as above.
The compound Y(R2)p can be chosen from the group: ammonia, mono-di- or poly-alkylamines, mono-di- or poly-alkylalcohols, mono-di- or poly-alkylthiols, with the amine-, alcohol-, or thiol group respectively being a primary group. The alkyl group can be linear or branched and the length of the alkyl group is preferably not more than C18 in order to comply with solubility requirements for the preferred solvents. The longer the alkyl group is the more hydrophobic the chromatographic media will be. Longer alkyl groups will also result in a reduction of small size pores ( less than 50 xc3x85 in diameter) of the polymer article.
In the second step an aqueous suspension of the brominated articles is mixed with e.g. the amine. It is also possible to use a mixture of water and a lower alcohol like methanol or ethanol as suspension medium. The reaction temperature is suitably 50-100xc2x0 C. and the reaction time about 24 h.
If the brominated articles are reacted with ammonia or a primary amine (mono, di or poly), coated polymer articles having the structure: 
or a mixture of structures: (5) or a mixture of structures: (5) or 
or the structure: 
or a mixture of structures: (16) and 
are obtained, where R1, R2, R3 and q have the same meaning as above. These compounds can be further reacted with an acid chloride, acid anhydride or isocyanate: 
or whereby a coated polymer article having the structure 
or a mixture of the structures: (9) and 
or having the structure: 
or a mixture of the structures: (11) and 
or a coated polymer article having the structure: 
or a mixture of the structures: (18) and 
or the structure: 
or a mixture of structures: (20) and 
are obtained where R1, R2, R3, and R4 and q have the same meaning as above and Z is halogen, such as chloride.
The reaction of the amine substituents with an acid chloride, acid anhydride or isocyanate is made in a water free organic solvent such as acetone, or another suitable industrial ketone. The solvent must not contain groups capable of reacting with the acid chloride/anhydride or isocyanate. It is possible to use water if the temperature is kept below 15xc2x0 C. During this reaction HCl is produced if an acid chloride is used. HCl can be neutralised with a tertiary amine, like di-isopropyl-ethylamine. The reaction is carried out at a temperature of 20-50xc2x0 C. and the reaction time suitably 1-3 h.
As an alternative to this last reaction step and if the substituent R1 is H, both the amine substituent and the hydroxyl group of formulae 5, 6, 16 and 17 may be reacted with an acid chloride, an anhydride or an isocyanate whereby a coated polymer article having the structure: 
or a mixture of the structure: (14) and (10) or having the structure 
or a mixture of structures: (15) and (12) or having the structure: 
or a mixture of structures: (23) and (19), or having the structure: 
or having a mixture of the structures: (24) and (21) are obtained.
The double bonds of the pending groups may also be converted to epoxide groups or corresponding sulphur or amino analogous by techniques well known in the art. For halohydrins or vicinal dihalides a proper adjustment of pH may result in epoxides, by doing this in the presence of the appropriate sulphur and amino compounds, the respective sulphur and nitrogen analogue will form. These three-membered rings will then be prone to undergo nucleophilic attack by the same compounds as outlined above for halohydrins and vicinal dihalides. The resulting products may then be further derivatized, also as outlined above. An alternative for obtaining epoxides is to react the double bond with a peroxy compound.
Further the introduced groups (coat) may then be further derivatized for instance to introduce charged groups such as xe2x80x94SO3xe2x88x92 (sulphonate), xe2x80x94PO32xe2x88x92 (phospohonate), xe2x80x94Oxe2x80x94PO32xe2x88x92 (phosphate), charged ammonium groups (primary, secondary, tertiary and quaternary ammonium groups) (free valences bind to saturated or unsaturated carbon).
The coated polymer articles can be used as chromatography media of different types. By varying the grade of halogenation, for instance bromination, the type and amount of e.g. amine and the type and amount of acid halide, such as acid chloride, or isocyanate it is possible to manufacture particles with a great variety in surface characteristics. Thus, with the invention it is possible to tailor the surface characteristics for the different molecules that are to be chromatographically separated. According to a preferred embodiment of the invention the coated articles are used as RPC media.
The introduction of primary or secondary amino groups will enable that the inventive article can be used in the solid phase synthesis of oligonucleotides and oligopeptides.